To accurately segregate its chromosomes, a dividing cell must first assemble a mitotic spindle, of which the main structural elements are microtubules. During spindle assembly, these dynamic cytoskeletal polymers are organized in space and time by microtubule-based motors and other molecular forces, ultimately giving rise to a steady-state structure with a characteristic spindle-like shape. Thus, spindle assembly is fundamentally a biomechanical phenomenon, and while numerous studies have focused on the underlying molecular mechanisms, little is known regarding the underlying mechanics. Addressing this gap in our understanding will require direct physical perturbations and quantitative measurements of the forces generated during spindle assembly. We propose to develop an innovative approach to the manipulation and analysis of mitotic spindle assemblies. Specifically, we will develop and apply a microfluidic-based platform for encapsulating nuclei within aqueous droplets that are suspended in a continuous mineral oil phase. This approach will allow us to investigate how physical constraints imposed by limited cytoplasmic volume impact bipolar spindle assembly. This question regarding the relationship between cytoplasmic volume and spindle scaling is fundamental but, until now, unanswered. Our techniques, however, will allow us to precisely regulate confinement and thus quantify forces during spindle assembly and study inherent mechanisms of spindle pole coalescence during bipolarization. This work serves our ultimate ambitions, which are to better understand the remarkable fidelity of chromosome segregation by elucidating the biomechanics that govern bipolar spindle assembly and function. Because errors in this process can lead to aneuploidy, a hallmark of neoplastic transformation and the cause of chromosomal birth defects, filling this gap has important implications in the context of human disease. To these ends, the Specific Aims of this proposal are: 1) To analyze the relationship between cytoplasmic volume and spindle size using well-defined extract-mineral oil emulsions, and 2) To develop and apply a high-throughput microfluidic-based approach to regulate spindle:droplet ratio and confinement for in vitro analysis of spindle pole coalescence. PUBLIC HEALTH RELEVANCE: In order to accurately segregate its chromosomes, a dividing cell must first assemble a mitotic spindle, which is fundamentally a biomechanical phenomena. Despite the remarkable fidelity with which this process is conducted, errors do occur that can lead to aneuploidy, a hallmark of neoplastic transformation and the cause of chromosomal birth defects. As such, developing a understanding of the biomechanical process by which nuclei replicate has profoundly important implications in the context of human disease. In this project we propose the development of a microfluidic experimental platform that will allow us to encapsulate nuclei and, in turn, establish relationships between the physical environment and the biomechanics of spindle assembly. This information may be used to develop more accurate and predictive models of spindle assembly, which in turn may lead to new strategies for the treatment of cancers and other diseases linked to improper spindle function.